PROJECT SUMMARY Antibodies are highly-specific, diverse and widely-assayed biomarkers used to determine recent or historical pathogen exposures, measure the protection conferred by a vaccine, understand the basis of autoimmune diseases or evaluate a host's immunological function. Traditional assays for antibodies focus on one or a small number of reactivities at a time, and so are incommensurate with the scale and diversity of an individual's antibody response. A tool to more holistically interrogate this diversity of reactivities using a small sample volume would enable a new generation of studies in systems immunology, disease association, and epidemiological surveillance. Here, we propose to optimize and significantly extend an approach we have developed for highly- multiplexed, reproducible and inexpensive assays that enable sensitive and high-resolution analysis of antibody reactivity across 100,000s of antigens from <1L of blood. Our approach takes advantage of a rapid, fully-in- vitro method for generating 100,000s of DNA-barcoded peptides (`PepSeq') as probes for the highly-multiplexed interrogation of serum antibodies using DNA sequencing. As a proof-of-concept, we will be focusing here on an assay targeting all viruses known to infect humans (i.e., the human virome). The virome is an ideal use case for this technology, as viruses represent an incredibly diverse and ubiquitous challenge to the immune system, and because of their small genome sizes, the complete virome can be covered within a single library with minimal loss of diversity. Our preliminary data with this virome assay establishes the feasibility of this approach. Here, we will optimize the assay procedures for multiple sample types in order to increase sensitivity and specificity, while decreasing cost. We will also establish standardized protocols for isotype-specific profiling, adapt the technology to enable antigen-specific, single-cell characterization, and build a suite of open access data analysis and visualization tools to facilitate the use of this technology by the broader research community. Throughout this process, we will generate a panel of anti-virome antibody profiles, including a cohort profiled longitudinally ? this data will be made available to the community through the ImmPort portal. If successful, this project will deliver: (i) an optimized assay SOP and library for comprehensive evaluation of pan-viral immunity using a small sample volume, (ii) a large set of publicly-available anti-virome immunity datasets, and (iii) a framework for multiplexed serological assay development that can be directly extended to other targets.